Activation of FGF receptors by mutations in the transmembrane domain

Abstract

Signaling through FGF receptors, which constitute a family of membrane-spanning tyrosine kinases, can stimulate cell proliferation, induce or inhibit cell di erentiation and plays an important role in development. Recently, mutations in FGF receptors have been shown to be associated with a number of genetically dominant human skeletal disorders. A remarkably conserved mutation (Gly 380?Arg) in the transmembrane region of FGFR-3 has been shown to be responsible for achondroplasia (ACH) but it was not clear whether such mutations result in loss of receptor function or constitutive activation. We have therefore made mutations in the transmembrane regions of murine FGFR-2 and FGFR-3 and studied their e ect on receptor activity. We show here that the ACH mutation in FGFR-3 as well as two similar mutations in FGFR-2 result in constitutive activation of these receptors. This is manifested in their ability to become autophosphorylated in the absence of ligand in L6 cells, transforming activity on NIH3T3 ®broblasts, and the ability to inhibit myogenic di erentiation in the absence of growth factor. Thus the transmembrane region of FGFR-2 and FGFR-3 plays a regulatory role in receptor function and the ACH mutation produces a dominant oversignaling receptor which is no longer regulated by FGF binding. These ®ndings also support the newly identi®ed role of FGF signaling as a negative regulator of bone growth.

Deregulated FGFR3 mutants in multiple myeloma cell lines with t(4;14): comparative analysis of Y373C, K650E and the novel G384D mutations

“…Our data indicate that deregulated FGFR3 mutants in KMS-11 and OPM-2 cells are phosphorylated under serum-deprived conditions, thus con®rming that these two mutations are capable of inducing constitutive receptor activation as has been observed in various cell types transfected with FGFR3 constructs (Naski et al, 1996;Webster et al, 1996;Webster and Donoghue, 1997b;Li et al, 1997). These data are supported by our analysis of U266 transfected cells expressing wildtype or Y373C FGFR3, which showed that only the mutated receptor is phosphorylated in the absence of the ligand.…”

FGF signaling activates STAT1 and p21 and inhibits the estrogen response and proliferation of MCF-7 cells

“…The activated receptor has a cysteine to arginine mutation at position 382 in the transmembrane domain. We have previously shown that this receptor is capable of transforming NIH3T3 cells and is tyrosine-phosphorylated even in the absence of ligand (Li et al, 1997). Several clones were isolated and those expressing the highest levels of FGFR-2/ C382R were selected for further study.…”

“…Expression of Bek 2.0 kb considerably decreased the FGFinhibition of CAT activity in the MCF-7 cells. Expression of a construct encoding the wild type FGFR-2 reduced overall CAT activity, presumably due to receptor activation resulting from high levels of expression (Li et al, 1997), but FGF-mediated inhibition of the estrogen response was still detectable. Cotransfection with a plasmid encoding the activated FGFR-2/C382R strikingly reduced the activity even further although estrogen-inducibility was still maintained.…”

“…NIH3T3 cells are a mouse ®broblast cell line grown in DMEM supplemented with 10% calf serum. 1021 #18 cells were obtained by calcium phosphate transfection of MCF7 cells with a plasmid containing the activated murine FGFR-2 mutant C382R in the pRK5 vector (Li et al, 1997), and selection by neomycin resistance. 1021 #18 cells were maintained in DMEM+10% FCS+400 mg/ml Geneticin (Life Technologies, Grand Island, NY).…”

FGF signaling pathways in endochondral and intramembranous bone development and human genetic disease

“…This mutation activates FGFR3 in the absence of ligand. However, the mutant receptor is further activated in the presence of ligand (Naski et al 1996;Webster and Donoghue 1996;Li et al 1997). Two different substitution mutations account for most cases of TD.…”